It is difficult to comprehend the affects of a smallpox outbreak. But the possibility exists, as remote as it may be. One line of control is the use of antiviral compounds. DNA replication enzymes are validated targets of antiviral compounds. This proposal is an intensive biochemical characterization of the proteins involved in replication of the poxvirus genome. The vaccinia virus, a close relative of smallpox, has duplex DNA genome that replicates entirely in the cytoplasm and encodes most or all of its own DNA replication proteins. Among these are the E9 DNA polymerase, A20 polymerase accessory protein, and the D4 uracyl deglycosylase (UDG) polymerase accessory proteins. These three proteins, E9/A20/D4 constitute a processive E9 holoenzyme replicase. In addition the virus encodes the essential D5 NTPase, Bl kinase and a single strand DNA binding protein called 13. We have recently purified recombinant E9 and A20/D4 complex and have constituted the E9 holoenzyme which forms the preliminary data for this proposal. We plan to determine the contact points between these proteins and understand how they provide a tight yet mobile grip of the E9 holoenzyme to DNA. We will also determine if the D5 NTPase is a helicase, and whether it functionally interacts with E9 holoenzyme. Alternatively, D5 may supply some other function to the replicative machinery. We will also examine the role(s) of the 13 SSB in D5 and E9 holoenzyme action. Finally, Rockefeller University's 20,000 chemical compound library will be screened for inhibitors of the E9 holoenzyme as a proof of principle that the proteins and assays developed herein can be useful to a pharmaceutical company with the proper tools and expertise to develop an antiviral compound.